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Regular Article
MYELOID NEOPLASIA
Mutations in the cohesin complex in acute myeloid leukemia: clinical
and prognostic implications
Felicitas Thol,1 Robin Bollin,1 Marten Gehlhaar,1 Carolin Walter,2 Martin Dugas,2 Karl Josef Suchanek,1 Aylin Kirchner,1
Liu Huang,1,3 Anuhar Chaturvedi,1 Martin Wichmann,1 Lutz Wiehlmann,4 Rabia Shahswar,1 Frederik Damm,5
Gudrun Göhring,6 Brigitte Schlegelberger,6 Richard Schlenk,7 Konstanze Döhner,7 Hartmut Döhner,7 Jürgen Krauter,1,8
Arnold Ganser,1 and Michael Heuser1
1
Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 2Institute of Medical
Informatics, Münster University, Münster, Germany; 3Department of Oncology, Tongji Hospital, Tongji Medical College, University of Science and
Technology, Huazhong, China; 4Clinical Research Group, Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School,
Hannover, Germany; 5Department of Hematology, Oncology, and Tumor Immunology, Charité, Berlin, Germany; 6Institute of Cell and Molecular Pathology,
Hannover Medical School, Hannover, Germany; 7Department of Internal Medicine III, University of Ulm, Ulm, Germany; and 8Klinikum Braunschweig,
Braunschweig, Germany
Mutations in the cohesin complex are novel, genetic lesions in acute myeloid leukemia
(AML) that are not well characterized. In this study, we analyzed the frequency, clinical,
and prognostic implications of mutations in STAG1, STAG2, SMC1A, SMC3, and RAD21,
• Mutations in genes of
all members of the cohesin complex, in a cohort of 389 uniformly treated AML patients by
the cohesin complex are
next generation sequencing. We identified a total of 23 patients (5.9%) with somatic
recurrent mutations in AML
with a strong association with mutations in 1 of the cohesin genes. All gene mutations were mutually exclusive, and
STAG1 (1.8%), STAG2 (1.3%), and SMC3 (1.3%) were most frequently mutated. Patients
NPM1 mutations.
with any cohesin complex mutation had lower BAALC expression levels. We found
• Cohesin gene mutations have
a strong association between mutations affecting the cohesin complex and NPM1.
no clear prognostic impact in
Mutated allele frequencies were similar between NPM1 and cohesin gene mutations.
AML patients.
Overall survival (OS), relapse-free survival (RFS), and complete remission rates (CR) were
not influenced by the presence of cohesin mutations (OS: hazard ratio [HR] 0.98; 95%
confidence interval [CI], 0.56-1.72 [P 5 .94]; RFS: HR 0.7; 95% CI, 0.36-1.38 [P 5 .3]; CR: mutated 83% vs wild-type 76% [P 5 .45]). The
cohesin complex presents a novel pathway affected by recurrent mutations in AML. This study is registered at www.clinicaltrials.gov
as #NCT00209833. (Blood. 2014;123(6):914-920)
Key Points
Introduction
Over the last several years, our knowledge of genes being mutated in
acute myeloid leukemia (AML) patients has not only expanded due to
the results of next generation and whole genome sequencing efforts,
but we have also learned that mutations in AML occur in specific
pathways. These pathways can be categorized according to their
function.1,2 In the last year, genes in the cohesin complex have been
described as novel mutations occurring in 13% of AML patients,1
suggesting that the cohesin-complex presents an important pathway in the pathogenesis of AML. Genes that belong to the cohesin
complex in somatic vertebrate cells are SMC1A, SMC3, RAD21
(SCC1), STAG2 (SA-2), and STAG1 (SA-1)3; these genes form a ring
structure that regulates chromosome segregation during meiosis and
mitosis. Thus, the cohesin-complex is an essential structure during cell
division.4 Undoubtedly, cell division is one of the key processes for
every tumor cell including AML blasts due to the increased proliferation
potential of malignant cells. Interestingly, more recent data suggests that
cohesin genes have additional functions within the cell such as doublestrand DNA repair and regulation of transcription. 5 It is known
that germline mutations lead to cohesinopathies that are characterized by growth and developmental disorders in regard to the role of
cohesin genes in the pathogenesis of human disease.6 Mutations in
the cohesin-complex have already been described in colorectal
cancer, and there has been a link between these mutations and chromosomal instability.7 Further investigation is needed to determine
which role the cohesin-complex fulfills in AML and whether cohesin
mutations have clinical implications. The aim of this study was to
investigate the frequency, clinical implications, and prognostic influence of mutations in the cohesin complex in the context of other
prognostic markers in a cohort of 389 uniformly treated AML patients.
Submitted July 31, 2013; accepted November 26, 2013. Prepublished online
as Blood First Edition paper, December 13, 2013; DOI 10.1182/blood-201307-518746.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2014 by The American Society of Hematology
914
Patients, materials, and methods
Patients
Diagnostic bone marrow or peripheral blood samples were analyzed from 389
adult patients (aged 17-60 years) with de novo (n 5 348) or secondary AML
BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
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BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
(n 5 41, and of these, 35 patients had antecedent myelodysplastic syndrome
and 6 patients had treatment-related secondary AML) with FrenchAmerican-British classification M0-M2 or M4-M7. These patients were
entered into the multicenter treatment trial AML SHG 0199 (#NCT00209833,
June 1999 to September 2004, n 5 276) or AML SHG 0295 (February 1995 to
May 1999, n 5 113) for whom pretreatment cell samples were available.
Patients with PML-RARA or t(15;17)-positive AML were excluded from these
trials. All patients received intensive, response-adapted double induction and
consolidation therapy. Details of the treatment protocols have been previously
reported.8,9 Peripheral blood mononuclear cells from 30 healthy volunteers
were used to assess the frequency of germline single nucleotide variants
(SNVs). Written informed consent was obtained according to the Declaration of
Helsinki, and the studies were approved by the institutional review board of
Hannover Medical School, Hannover, Germany.
Cytogenetic and molecular analysis
Pretreatment samples from all patients were studied centrally by G- and
R-banding analysis. Chromosomal abnormalities were described according
to the International System for Human Cytogenetic Nomenclature.10 Other
relevant genes were assessed for frequently occurring mutations or expression
levels as previously described (ie, FLT3-ITD,8 nucleophosmin1 [NPM1],8
DNMT3A,11 IDH1,12 IDH2,13 and MLL514). In the subgroup of cytogenetically normal AML (CN-AML), additional mutation analyses were performed
for CEBPA,15 MLL-PTD,16 WT1, and WT1 SNP rs16754,17 NRAS,8 and
expression levels of BAALC,18 ERG,19 EVI1,20,21 MN1,22 MLL5,14,17 and WT117
were quantified as previously described using complementary DNA from the
KG1A cell line (BAALC, ERG, MLL5), plasmids (MN1,23 WT117), or from
a patient sample (EVI1) to construct a relative standard curve using ABL as
a housekeeping gene (Ipsogen, Marseille, France).
COHESIN GENE MUTATIONS IN AML
915
(CD31 CD11b2 CD142 CD332 ) purified from diagnostic samples by flow
cytometry.
Statistical analysis
The definition of complete remission (CR), overall survival (OS), and relapsefree survival (RFS) followed recommended criteria.27 Primary analysis was
performed on OS. Sensitivity analyses were performed on CR and RFS, and
results are displayed for exploratory purposes. Median follow-up time for
survival was calculated according to the method of Korn.28 OS endpoints
measured from the date of entry into one of the prospective studies were death
(failure) and alive at last follow-up (censored). RFS endpoints measured from
the date of documented CR were relapse (failure), death in CR (failure), and
alive in CR at last follow-up (censored).
Pairwise comparisons of variables were performed for exploratory purposes using the Kolmogorov-Smirnov test and Student t test for continuous
variables and the x-squared test for categorical variables. The Kaplan-Meier
method and log-rank test were used to estimate the distribution of OS and RFS,
and to compare differences between survival curves, respectively. Mutations
in the analyzed genes were used as categorical variables. To determine the
expression levels of EVI1, relative quantification was calculated using the
equation 22DDCt, as previously described.20 To provide quantitative information on the relevance of results, 95% confidence intervals (CIs) of odds
ratios (OR) and hazard ratios (HR) were computed. Two-sided P values ,.05
were considered significant in the primary analysis and indicators for a trend
in all additional analyses. Statistical analyses were performed with the statistical
software package SPSS 20.0 (IBM Corp., Armonk, NY). Associations
between gene mutations are represented by a Circos diagram (Figure 1).29
Analysis of cohesin mutations
Leukemic cells from peripheral blood or bone marrow were collected from
patients at diagnosis, and genomic DNA was extracted, whole genome
amplified (GenomePlex whole genome amplification kit, Sigma-Aldrich,
Seelze, Germany) and polymerase chain reaction amplified for 119 amplicons
with not more than 5 amplicons per well using standard conditions. The
primers for all exons of the cohesin genes are listed in supplemental Table 1,
available on the Blood Web site. All amplicons from 1 patient were pooled
and randomly ligated (Quick Ligation Kit, New England Biolabs, Ipswich,
MA). The long concatenated DNA was then sheared into 100 to 250 bp
fragments using the Covaris System (Covaris, Woburn, MA) to obtain
randomly fragmented sequences, and was size selected for 200 bp fragments
using Agencourt AMPure XP reagent (Agencourt Bioscience Corp., Beverly,
MA). Patient-specific barcodes and sequencing primers P1 and P2 were ligated
to these fragments. The fragments were loaded and amplified with the SOLiD
sequencing control beads during emulsion polymerase chain reaction. The beads
were then added to the Flow Chip for sequencing in the SOLiD system (Life
Technologies, Darmstadt, Germany), and sequenced according to the
manufacturer’s protocol (Life Technologies). Individual reads were 75 bp
long. Reads were assigned to their patient-specific barcode, and sequences
were analyzed twice separately using the 2010 DNAnexus software and
the following pipeline of bioinformatics software. The color-space reads
were aligned with NovoalignCS24 and genotyped with GATK’s Unified
Genotyper.25 SNV and indel discovery was performed across all samples
using standard parameters and a maximum coverage of 10 000. The mean
coverage of all amplicons was 2484 reads per amplicon. The resulting list of
candidate SNVs was filtered with R.23 First, mutations outside the coding region
were excluded. Second, known single nucleotide polymorphisms (SNPs) were
removed (dbSNP, version 137). Third, mutations with a quality score of ,8000
or an allele frequency of ,15% were excluded. The remaining mutations were
validated by Sanger sequencing.
To determine the coverage of genomic intervals, the data were processed
with BEDTools26 and analyzed in R. Mutations were validated by Sanger
sequencing and are only reported if they were detected by Sanger sequencing
either in genomic DNA or in an independently whole genome-amplified
DNA sample. The somatic or germline status of mutations in genes of the
cohesin complex was established by evaluating remission samples or T cells
Results
Mutations in the cohesin gene complex
In our cohort, we identified mutations in genes of the cohesin
complex in 23 AML patients (5.9%). The most commonly mutated
gene in this complex was STAG1 with 7 patients harboring a mutation
in this gene. The second most frequently mutated genes in the cohesin
complex were SMC3 and STAG2, and we found 5 patients with these
mutations, respectively. Additionally, we identified 4 patients with
mutations in RAD21 and 2 patients with mutations in SMC1A
(Figure 1). Interestingly, all mutations were mutually exclusive
among each other. No mutation hotspot was identified in any of the
genes (Figure 1 and supplemental Tables 2-6). STAG2 and SMC1A are
X-linked, whereas the other 3 genes are autosomal. Of the 5 patients
with STAG2 mutations, 2 patients were female and 3 were male. Both
patients with SMC1A mutations were male. The male patients had a
functionally homozygous mutation for STAG2 and SMC1A.
Six of 7 mutations in STAG1 were missense mutations, while one
patient showed a frameshift mutation (Figure 1 and supplemental
Table 2). Two patients with mutations in STAG2 had missense
mutations, 2 patients had frameshift mutations, and 1 patient had
a nonsense mutation in this gene (Figure 1 and supplemental
Table 3). Four patients were identified with missense mutations in
SMC3 and 1 patient with the stop codon being changed to leucine
(Figure 1 and supplemental Table 4). In RAD21, we found 2 patients
with missense mutations, 1 patient with a frameshift mutation, and 1
with a nonsense mutation (Figure 1 and supplemental Table 5). The 2
mutations in SMC1A were both missense mutations (Figure 1 and
supplemental Table 6). Only the mutation in R381Q of SMC3 was
recurrent in 2 patients, whereas all other mutations were only
identified once (Figure 1 and supplemental Table 4).
From a total of 23 putative mutations in cohesin genes, the
somatic status could be confirmed in 16 by analyzing remission
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916
THOL et al
BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
Figure 1. Location and type of mutations in genes of the cohesin complex in 389 patients with AML, and associations of gene mutations in the AML patient cohort
outlined by a Circos diagram.
samples or T-cells (CD31 CD11b2 CD142 CD33), which were
purified from diagnostic samples by flow cytometry (supplemental
Tables 2-6). For the remaining patients with mutations, the somatic
origin could not be confirmed due to lack of suitable material.
Besides these somatic mutations in the cohesin complex, in our
analysis, we also identified 3 unannotated SNPs (supplemental
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BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
COHESIN GENE MUTATIONS IN AML
Table 1. Comparison of pretreatment characteristics between
patients with and without mutations in the cohesin complex genes
Cohesin genes
mutated (n 5 23)
no. (%)
Cohesin genes
wild-type (n 5 366)
no. (%)
Median
46
44.5
Range
28-60
17-60
Characteristic
Age, years
.13
Sex
13 (57%)
185 (51%)
Female
10 (43%)
181 (49%)
ECOG performance status
.37
Cohesin genes
mutated (n 5 23)
no. (%)
Cohesin genes
wild-type (n 5 366)
no. (%)
IDH2
1 (4%)
30 (8%)
Wild-type
20 (87%)
271 (74%)
2 (9%)
65 (18%)
Mutated
1 (4%)
56 (15%)
Wild-type
20 (87%)
244 (67%)
2 (9%)
66 (18%)
IDH1/2
Missing
.11
0
5 (22%)
90 (25%)
1
16 (70%)
221 (60%)
Mutated
6 (26%)
68 (19%)
2
1 (4%)
53 (14%)
Wild-type
17 (74%)
292 (80%)
Missing data
1 (4%)
2 (1%)
0 (0%)
6 (2%)
FAB-subtype
DNMT3A
Missing
.53
.4
NRAS
.48
M0
0 (0%)
7 (2%)
Mutated
2 (9%)
50 (14%)
M1
8 (35%)
59 (16%)
Wild-type
18 (78%)
263 (72%)
M2
5 (22%)
90 (25%)
Missing
3 (13%)
53 (14%)
M4
7 (30%)
111 (30%)
M5
2 (9%)
56 (15%)
9.03
15.15
M6
0 (0%)
6 (2%)
M7
0 (0%)
3 (1%)
0.31-31.7
0.62-5134.5
Missing data
1 (4%)
17 (5%)
1.70
4.70
0.04-26.2
0.026-806.4
28.1
33.4
0.67-125.3
0.07-1605.7
0 (0%)
6 (2%)
Type of AML
MN1
Median copy number
Secondary
BAALC
Favorable
326 (89%)
(relative BAALC/ABL)
1 (4%)
40 (11%)
Range (relative BAALC/
.26
1 (4%)
61 (17%)
17 (74%)
257 (70%)
Adverse
4 (17%)
44 (12%)
Missing data
1 (4%)
4 (1%)
Intermediate
Peripheral blood blasts
Mean
Missing data
58.1
46.7
1 (4%)
15 (4%)
74.1
71.4
Missing data
0 (0%)
24 (7%)
Median (x109/L)
26.6
21.6
1.3-229
0.5-328
Missing data
0 (0%)
1 (0%)
Median (g/L)
8.4
9
Range (g/L)
5.5-11.8
3-15.4
Missing data
0 (0%)
9 (2%)
Hemoglobin
Expressers
9
39
Missing
14 (61%)
157 (43%)
9 (39%)
203 (55%)
34.3
38.2
14.04-174.5
4.45-498
0.87
1.2
0.035-5.88
0.001-231.3
MLL5
Median copy number
.85
(relative MLL5/ABL)
Range (relative MLL5/
ABL)
WT1
Median copy number
.29
(relative WT1/ABL)
Range (relative WT1/ABL)
48.5
Range – (x10 /L)
7-215
2.6-483
Missing data
0 (0%)
10 (3%)
Mutated
6 (26%)
98 (27%)
Wild-type
17 (74%)
266 (73%)
0 (0%)
2 (1%)
Mutated
13 (57%)
124 (34%)
Wild-type
10 (43%)
240 (66%)
0 (0%)
2 (1%)
FLT3-ITD
Missing
.93
NPM1
Missing
.029
NPM1/FLT3 mutation risk
.024
group
Low risk*
9 (39%)
71 (19%)
High risk*
14 (61%)
293 (80%)
0 (0%)
2 (1%)
Missing
IDH1
R132 mutated
Missing
Nonexpressers
.47
.26
Median – (x109/L)
Wild-type
ABL)
EVI1
.09
Platelet count
.64
(relative ERG/ABL)
Range (relative ERG/
.66
Range (x109/L)
ABL)
Median copy number
.6
WBC count
.033
ERG
.1
Bone marrow blasts
Mean
Median copy number
22 (96%)
Cytogenetic risk group†
.071
(relative MN1/ABL)
Range (relative MN1/ABL)
.32
De novo
P
.43
Mutated
Missing
.58
Male
Table 1. (continued)
Characteristic
P
917
.15
0 (0%)
26 (7%)
22 (96%)
276 (75%)
1 (4%)
64 (17%)
ECOG, performance status of the Eastern Cooperative Oncology Group; FAB,
French-American-British classification of AML; FLT3-ITD, internal tandem duplication
of the FLT3 gene; no., number; P, P value from two-sided x-squared tests for categorical
variables and from 2-sided Student t or Kolmogorov-Smirnov tests for continuous
variables.
*The high-risk molecular group is defined as either NPM1wild-type/FLT3-ITDnegative,
or NPM1 wild-type/FLT3-ITD positive, or NPM1mutated/FLT3-ITD positive. The low-risk molecular group is defined by the presence of an NPM1 mutation and the absence of FLT3-ITD.
†The cytogenetic risk group is defined according to Medical Research Council
criteria.30
Table 7). These SNPs have not been reported in dbSNP (version 137)
and were not identified in peripheral blood mononuclear cells from
30 healthy controls.
Association of cohesin gene mutations with clinical
characteristics
Most clinical and disease characteristics of patients with mutations in
the cohesin complex were similarly distributed, as in patients with
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918
BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
THOL et al
Table 2. Univariate analysis for OS and RFS in AML patients
(n 5 389) according to cohesin gene mutation status
OS
Endpoint
RFS
HR
95% CI
P
HR
95% CI
P
0.98
0.56-1.72
.94
0.70
0.36-1.38
.3
STAG1 mutated vs wild-type
0.68
0.22-2.12
.5
0.50
0.12-2
.33
STAG2 mutated vs wild-type
1.53
0.57-4.11
.4
1.26
0.4-3.93
.7
SMC3 mutated vs wild-type
0.28
0.04-1.97
.2
0.05
0-4.75
.19
RAD21 mutated vs wild-type
1.47
0.47-4.60
.51
1.02
0.25-4.1
.98
Cohesin mutations combined
Cohesin mutated vs wild-type
Cohesin mutations separately
HR .1 or ,1 indicate an increased or decreased risk, respectively, of an event
for the first category listed.
P, P value from univariate or multivariate Cox proportional hazards models.
wild-type cohesin genes (Table 1).30 Only 1 patient with favorable
cytogenetics had a mutation in a cohesin gene (SMC1A), whereas the
majority of patients with cohesin gene mutations had intermediate risk
cytogenetics, most showing a normal karyotype (supplemental Tables
1 and 2-6). Interestingly, we found a strong correlation between the
cohesin gene and NPM1 mutations (57% of cohesin gene mutated
patients had an NPM1 mutation; 9.5% of NPM1 mutated patients had
a cohesin gene mutation compared with 4% of NPM1 wild-type
patients; P 5 .029) (Table 1). Of 7 patients with STAG1 mutations, 3
patients also showed an NPM1 mutation. Of 5 STAG2 and SMC3
mutated patients, 3 carried a mutation in NPM1, respectively. Of the
4 RAD21 mutated patients, 3 harbored a concomitant NPM1 mutation,
and 1 of the 2 SMC1 mutated patients was also NPM1 mutated
(supplemental Tables 2-6). No correlation was observed between
genes in the cohesin complex and other mutations such as FLT3-ITD,
IDH1, IDH2, or NRAS mutations. BAALC expression was lower in
patients with a mutation compared with patients without a mutation
in a gene of the cohesin complex (P 5 .033) (Table 1). There was no
significant difference between patients with or without a mutation in
the cohesin complex with regard to gene expression of MN1, ERG,
EVI1, MLL5, and WT1. To get a better understanding of when mutations
in the cohesin complex occur during clonal evolution, we evaluated the
allelic burden of mutations in the cohesin complex. Because of
the strong association between NPM1 and cohesin mutations, the
allelic ratio of mutated and wild-type NPM1 was compared with
the allelic ratio of mutated and wild-type cohesin genes. Interestingly, we
found a similar mutation burden between NPM1 and genes of the cohesin
complex in most patients (supplemental Figure 1), suggesting that
cohesin gene mutations occurred in the same clone as NPM1 mutations.
Clinical outcome in the total cohort of AML patients according
to cohesin gene mutation status
Median follow-up time for all patients was 5.1 years (range, 0.19 to
12.2 years). When considering all mutations in the cohesin complex
as 1 group, OS and RFS were not influenced by the presence of cohesin
mutations (OS: HR 0.98; 95% CI, 0.56-1.72; P 5 .94; Figure 2A)
(RFS: HR 0.70; 95% CI, 0.36-1.38; P 5 .3, Figure 2B and Table 2). In
addition, there was no difference between CR rates of mutated and
wild-type patients (mutated 83% vs wild-type 76%; P 5 .45). Similar
results were obtained when only considering patients with de novo
AML (n 5 348) with the exclusion of patients with secondary AML
(OS: HR 0.96; 95% CI, 0.54-1.73; P 5 .89; supplemental Figure 2A),
(RFS: HR 0.62; 95% CI, 0.3-1.25; P 5 .18; supplemental Figure 2B),
and (CR: mutated 82% vs wild-type 78%; P 5 .65). For exploratory
purposes, next we evaluated the prognostic influence of each gene
in the cohesin complex separately in all patients, although this analysis
is limited by the small number of mutated patients. STAG1, STAG2,
SMC3, and RAD21 mutations had no influence on OS and RFS,
whereas the analysis was not performed for SMC1A due to the low
mutation frequency (Table 2).
In the subgroup of patients with CN-AML (n 5 201), we identified
16 patients with mutations in the cohesin complex (8%). In this
subgroup, we did not identify a difference in OS and RFS for patients
with or without mutations in the cohesin complex (OS: HR 0.73; 95%
CI, 0.34-1.57; P 5 .42) and (RFS: HR 0.47; 95% CI, 0.19-1.16; P 5 .1;
supplemental Figure 3A-B). Again, no difference was found in CR
rates (mutated 88% vs wild-type 78%; P 5 .39). Due to the strong
association between NPM1 mutations and mutations in the cohesin
complex, we studied the impact of cohesin mutations on OS and RFS
in NPM1-mutated AML patients separately. In the group of NPM1mutated AML patients (n 5 137), OS and RFS were not influenced
by the presence of mutations in the cohesin complex (OS: HR 1.17;
95% CI, 0.53-2.55; P 5 .7; supplemental Figure 3C) and (RFS: HR
0.85; 95% CI, 0.34-2.12; P 5 .72; supplemental Figure 3D). When
considering the otherwise favorable prognostic group of patients with
mutated NPM1, but wild-type FLT3, no significant difference for OS
and RFS between patients with our without mutations in the cohesin
complex was identified (OS: HR 0.65; 95% CI, 0.2-2.14; P 5 .48;
supplemental Figure 3E) and (RFS: HR 0.32; 95% CI, 0.07-1.39;
P 5 .13; supplemental Figure 3F).
Similar results were obtained when considering NPM1-mutated
CN-AML patients or when looking at the different prognostic groups
in the European LeukemiaNet classification (data not shown).31
We analyzed the effect of allogeneic transplantation on OS of
patients with cohesin mutations. Our clinical trial protocol allowed
an intent-to-treat analysis on the basis of donor availability in patients
with CN-AML. The OS of cohesin-mutated patients with a related
donor was similar compared with patients without a related donor
(OS: HR 0.54; 95% CI, 0.06-4.8; P 5 .58; supplemental Figure 4A-B),
suggesting that cohesin gene mutations did not influence the outcome
of allogeneic transplantation.
Discussion
In this analysis of 389 well-characterized patients with AML, we
identified somatic mutations in the cohesin complex in 5.9% of
patients. In our cohort, STAG1 followed by STAG2 and SMC3
mutations were the most frequently mutated genes in the cohesin
complex, whereas SMC1A mutations were rare events. In the recent
report of the Cancer Genome Atlas Research Network, no mutations
in STAG1 were detected, whereas the mutation frequency in STAG2,
SMC1A, SMC3, and RAD21 was slightly higher (2.5%-3.5%).1 The
lower mutation frequency in our study may be explained by our
approach that we only considered mutations that could be validated
by Sanger sequencing and therefore are present in at least 10% to
20% of all cells. All mutations were heterozygous apart from STAG2
and SMC1A mutations in male patients, as both genes are X-linked.
Mutations occurred throughout the genes without the presence of
a mutational hotspot. Interestingly, mutations in this complex were
mutually exclusive, similar to other mutations that belonged to 1
pathway similar to genes of the spliceosome complex or mutations in
IDH1, IDH2, and TET2.13,32-34
To differentiate polymorphisms from somatic mutations, we
studied T-cells purified from diagnostic samples by flow cytometry.
In addition to the 23 somatic mutations, we identified 3 SNPs (2 in
STAG1 and 1 in RAD21), which have not been described by dbSNP
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COHESIN GENE MUTATIONS IN AML
919
Figure 2. Prognostic impact of cohesin mutations in all investigated AML patients. (A) OS in AML patients with wild-type (WT) or mutated genes of the cohesin
complex. (B) RFS in AML patients with WT or mutated genes of the cohesin complex.
and were not found in mononuclear cells of 30 healthy donors.
Although germline mutations in SMC1A, SMC3, and RAD21 have
been associated with cohesinopathies, pathogenic STAG1 germline
mutations have not been reported.35 All patients studied during
remission lost the mutation at this time point. This data implements
that cohesin mutations could be potentially used for minimal residual
disease monitoring. However, further studies are needed to analyze
the stability of these markers, as we also found 1 patient in which the
SMC1A mutation from the time of diagnosis was neither present
during remission nor at the time of relapse.
Most mutated patients had a normal karyotype, suggesting that
cohesin gene mutations do not act through destabilization of
chromosomal integrity in AML, but rather alternative mechanisms
such as transcriptional control. Interestingly, cohesin genes have been
found to bind to CCCTC-binding factor, a sequence-specific
transcription factor36 that is known to interact with NPM1 in addition
to regulating tumor suppressor loci.37 Importantly, cohesin genes can
also affect transcription independently of CCCTC-binding factor.38
We compared the allelic burden of mutations in the cohesin complex with the allelic burden of NPM1 mutations, as the level of the
allelic burden can indicate clonal hierarchy of different mutations.39
It has already been suggested that NPM1 is an early event in leukemogenesis.40 In our analysis, the allelelic burden of mutations in the
cohesin genes were very similar to that observed for NPM1 mutations.
This suggests that cohesin mutations may occur at a similarly early time
point of leukemogenesis.
An interesting finding was the strong association between NPM1
mutations and the mutations in cohesin genes, as already indicated in
the recent report of the Cancer Genome Atlas Research Network.1 In
our analysis, the prognostic implications of mutations in the cohesin
complex were not significant when considering all mutations in the
complex together or individually.
Because of the strong association between NPM1 mutations and
genes in the cohesin complex, we wanted to find out whether the
cohesin mutations have an impact in the NPM1-mutated patient
group. It could be possible that a negative prognostic impact
of cohesin mutations might have been missed because of the
favorable prognostic impact of NPM1 mutations and the strong
association between these mutations. However, in CN-AML patients,
in patients with mutated NPM1, and in the NPM1 mutated/FLT3
wild-type subgroup of patients, cohesin mutations had no impact
on outcome. Taken together, our data suggest that cohesin mutations
do not affect patient prognosis.
In summary, our results show that mutations in the cohesin
complex are recurrent mutations in AML. We found a strong association between these mutations and mutations in NPM1. Cohesin
complex mutations as a group had no prognostic impact in all and in
cytogenetically normal AML patients.
Acknowledgment
This study was supported by grants from Deutsche Krebshilfe
(109003, 110284, and 110292), a grant from the Deutsche-JoséCarreras Leukämie-Stiftung e.V. (DJCLS R 10/22), a grant from
the German Federal Ministry of Education and Research (01EO0802)
(IFB-Tx), and grants from the Deutsche Forschungsgemeinschaft
(HE 5240/4-1, HE 5240/5-1, and TH 1779/1-1).
Authorship
Contribution: F.T. and M.H. designed the research; F.T., R.B., M.G.,
K.J.S., A.K., L.H., A.C., M.W., LW., R.Shahswar, F.D., and M.H.
performed the research; F.T., R.Schlenk, K.D., H.D., J.K., A.G., and
M.H. contributed patient samples and clinical data; G.G. and B.S.
performed cytogenetic studies; F.T., C.W., M.D., A.G., and M.H.
analyzed the data; F.T., A.G., and M.H. wrote the paper; and all
authors read and agreed to the final version of the manuscript.
Conflict of interest disclosure: The authors declare no competing
financial interests.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
920
BLOOD, 6 FEBRUARY 2014 x VOLUME 123, NUMBER 6
THOL et al
Correspondence: Felicitas Thol, Department of Hematology,
Hemostasis, Oncology, and Stem Cell Transplantation, Hannover
Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany;
e-mail: [email protected].
References
1. Cancer Genome Atlas Research Network.
Genomic and epigenomic landscapes of adult de
novo acute myeloid leukemia. N Engl J Med.
2013;368(22):2059-2074.
2. Welch JS, Ley TJ, Link DC, et al. The origin and
evolution of mutations in acute myeloid leukemia.
Cell. 2012;150(2):264-278.
3. Remeseiro S, Losada A. Cohesin, a chromatin
engagement ring. Curr Opin Cell Biol. 2013;25(1):
63-71.
4. Wassmann K. Sister chromatid segregation in
meiosis II: deprotection through phosphorylation.
Cell Cycle. 2013;12(9):1352-1359.
5. Wendt KS, Yoshida K, Itoh T, et al. Cohesin
mediates transcriptional insulation by CCCTCbinding factor. Nature. 2008;451(7180):796-801.
16. Döhner K, Tobis K, Ulrich R, et al. Prognostic
significance of partial tandem duplications of the
MLL gene in adult patients 16 to 60 years old with
acute myeloid leukemia and normal cytogenetics:
a study of the Acute Myeloid Leukemia Study
Group Ulm. J Clin Oncol. 2002;20(15):3254-3261.
17. Damm F, Heuser M, Morgan M, et al. Single
nucleotide polymorphism in the mutational
hotspot of WT1 predicts a favorable outcome in
patients with cytogenetically normal acute myeloid
leukemia. J Clin Oncol. 2010;28(4):578-585.
18. Baldus CD, Martus P, Burmeister T, et al. Low
ERG and BAALC expression identifies a new
subgroup of adult acute T-lymphoblastic leukemia
with a highly favorable outcome. J Clin Oncol.
2007;25(24):3739-3745.
7. Barber TD, McManus K, Yuen KW, et al.
Chromatid cohesion defects may underlie
chromosome instability in human colorectal
cancers. Proc Natl Acad Sci USA. 2008;105(9):
3443-3448.
19. Marcucci G, Maharry K, Whitman SP, et al;
Cancer and Leukemia Group B Study. High
expression levels of the ETS-related gene, ERG,
predict adverse outcome and improve molecular
risk-based classification of cytogenetically normal
acute myeloid leukemia: a Cancer and Leukemia
Group B Study. J Clin Oncol. 2007;25(22):
3337-3343.
8. Schlenk RF, Döhner K, Krauter J, et al;
German-Austrian Acute Myeloid Leukemia Study
Group. Mutations and treatment outcome in
cytogenetically normal acute myeloid leukemia.
N Engl J Med. 2008;358(18):1909-1918.
20. Barjesteh van Waalwijk van Doorn-Khosrovani S,
Erpelinck C, van Putten WLJ, et al. High EVI1
expression predicts poor survival in acute myeloid
leukemia: a study of 319 de novo AML patients.
Blood. 2003;101(3):837-845.
9. Heil G, Krauter J, Raghavachar A, et al.
Risk-adapted induction and consolidation therapy
in adults with de novo AML aged ,/5 60 years:
results of a prospective multicenter trial. Ann
Hematol. 2004;83(6):336-344.
21. Damm F, Heuser M, Morgan M, et al. Integrative
prognostic risk score in acute myeloid leukemia
with normal karyotype. Blood. 2011;117(17):
4561-4568.
6. Gerton JL. Translational mechanisms at work in
the cohesinopathies. Nucleus. 2012;3(6):520-525.
10. Mitelman F. ISCN 1995: An International System
for Human Cytogenetic Nomenclature. Basel,
Switzerland: Karger; 1995.
11. Thol F, Damm F, Lüdeking A, et al. Incidence and
prognostic influence of DNMT3A mutations in
acute myeloid leukemia. J Clin Oncol. 2011;
29(21):2889-2896.
12. Wagner K, Damm F, Göhring G, et al. Impact
of IDH1 R132 mutations and an IDH1 single
nucleotide polymorphism in cytogenetically
normal acute myeloid leukemia: SNP rs11554137
is an adverse prognostic factor. J Clin Oncol.
2010;28(14):2356-2364.
13. Thol F, Damm F, Wagner K, et al. Prognostic
impact of IDH2 mutations in cytogenetically
normal acute myeloid leukemia. Blood. 2010;
116(4):614-616.
14. Damm F, Oberacker T, Thol F, et al. Prognostic
importance of histone methyltransferase MLL5
expression in acute myeloid leukemia. J Clin
Oncol. 2011;29(6):682-689.
15. Fröhling S, Schlenk RF, Stolze I, et al. CEBPA
mutations in younger adults with acute myeloid
leukemia and normal cytogenetics: prognostic
relevance and analysis of cooperating mutations.
J Clin Oncol. 2004;22(4):624-633.
22. Heuser M, Beutel G, Krauter J, et al. High
meningioma 1 (MN1) expression as a predictor for
poor outcome in acute myeloid leukemia with
normal cytogenetics. Blood. 2006;108(12):
3898-3905.
23. Heuser M, Argiropoulos B, Kuchenbauer F,
et al. MN1 overexpression induces acute myeloid
leukemia in mice and predicts ATRA resistance in
patients with AML. Blood. 2007;110(5):
1639-1647.
24. Novocraft Technologies. NovoalignCS. Available
at: www.novocraft.com. Accessed June 11, 2013.
25. DePristo MA, Banks E, Poplin R, et al. A
framework for variation discovery and genotyping
using next-generation DNA sequencing data. Nat
Genet. 2011;43(5):491-498.
26. Quinlan AR, Hall IM. BEDTools: a flexible
suite of utilities for comparing genomic features.
Bioinformatics. 2010;26(6):841-842.
27. Cheson BD, Bennett JM, Kopecky KJ, et al;
International Working Group for Diagnosis,
Standardization of Response Criteria, Treatment
Outcomes, and Reporting Standards for
Therapeutic Trials in Acute Myeloid Leukemia.
Revised recommendations of the International
Working Group for Diagnosis, Standardization of
Response Criteria, Treatment Outcomes, and
Reporting Standards for Therapeutic Trials in
Acute Myeloid Leukemia. J Clin Oncol. 2003;
21(24):4642-4649.
28. Korn EL. Censoring distributions as a measure
of follow-up in survival analysis. Stat Med. 1986;
5(3):255-260.
29. Krzywinski M, Schein J, Birol I, et al. Circos: an
information aesthetic for comparative genomics.
Genome Res. 2009;19(9):1639-1645.
30. Grimwade D, Walker H, Oliver F, et al; The
Medical Research Council Adult and Children’s
Leukaemia Working Parties. The importance of
diagnostic cytogenetics on outcome in AML:
analysis of 1,612 patients entered into the MRC
AML 10 trial. Blood. 1998;92(7):2322-2333.
31. Döhner H, Estey EH, Amadori S, et al; European
LeukemiaNet. Diagnosis and management
of acute myeloid leukemia in adults:
recommendations from an international expert
panel, on behalf of the European LeukemiaNet.
Blood. 2010;115(3):453-474.
32. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent
pathway mutations of splicing machinery in
myelodysplasia. Nature. 2011;478(7367):64-69.
33. Thol F, Kade S, Schlarmann C, et al. Frequency
and prognostic impact of mutations in SRSF2,
U2AF1, and ZRSR2 in patients with
myelodysplastic syndromes. Blood. 2012;
119(15):3578-3584.
34. Paschka P, Schlenk RF, Gaidzik VI, et al.
IDH1 and IDH2 mutations are frequent genetic
alterations in acute myeloid leukemia and confer
adverse prognosis in cytogenetically normal acute
myeloid leukemia with NPM1 mutation without
FLT3 internal tandem duplication. J Clin Oncol.
2010;28(22):3636-3643.
35. Barbero JL. Genetic basis of cohesinopathies.
Appl Clin Genet. 2013;6:15-23.
36. Lee BK, Iyer VR. Genome-wide studies of
CCCTC-binding factor (CTCF) and cohesin
provide insight into chromatin structure and
regulation. J Biol Chem. 2012;287(37):
30906-30913.
37. Zlatanova J, Caiafa P. CTCF and its protein
partners: divide and rule? J Cell Sci. 2009;
122(Pt 9):1275-1284.
38. Schmidt D, Schwalie PC, Ross-Innes CS, et al.
A CTCF-independent role for cohesin in tissuespecific transcription. Genome Res. 2010;20(5):
578-588.
39. Walter MJ, Shen D, Ding L, et al. Clonal
architecture of secondary acute myeloid
leukemia. N Engl J Med. 2012;366(12):
1090-1098.
40. Krönke J, Bullinger L, Teleanu V, et al. Clonal
evolution in relapsed NPM1-mutated acute
myeloid leukemia. Blood. 2013;122(1):100-108.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2014 123: 914-920
doi:10.1182/blood-2013-07-518746 originally published
online December 13, 2013
Mutations in the cohesin complex in acute myeloid leukemia: clinical
and prognostic implications
Felicitas Thol, Robin Bollin, Marten Gehlhaar, Carolin Walter, Martin Dugas, Karl Josef Suchanek,
Aylin Kirchner, Liu Huang, Anuhar Chaturvedi, Martin Wichmann, Lutz Wiehlmann, Rabia Shahswar,
Frederik Damm, Gudrun Göhring, Brigitte Schlegelberger, Richard Schlenk, Konstanze Döhner,
Hartmut Döhner, Jürgen Krauter, Arnold Ganser and Michael Heuser
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